Limbal stem-cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier-free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier-free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell-cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier-free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration.

The present study investigates the effect of afatinib on the growth, induction of apoptosis in RB116 cells, and reduction of carcinoma growth in the mice transplanted with RB116 cells. The results from MTT assay revealed that afatinib inhibited the growth of RB116 cells in a dose-dependent manner. Proliferation of RB116 cells was reduced to 64 % on treatment with 200 μM concentration of afatinib after 48 h. Afatinib treatment of RB116 cells at 200 μM concentration induced apoptosis and necrosis in 49.7 and 9.4 %, respectively, after 48 h. In the RB116-transplanted mice, treatment with afatinib at 10-mg/kg doses for 45 days caused a significant (p < 0.005) reduction in the tumor volume compared to the control group. The tissue lysates of the mice containing RB116 transplant showed a significant decrease in the expressions of Ki67 and p53 in the afatinib treatment group after 45 days. However, the expression of caspase-3 was increased and of Bcl-2 remained unaltered on treatment with afatinib. Measurement of the body weight of afatinib-treated animals showed no reduction during the study. Thus, afatinib can be of therapeutic value for the treatment of retinoblastoma.

ES culture supernatant (ESC-CM) regulates the fate of LSCs in part by inhibiting the miR-31/FIH-1/P21 axis. This study may have a high impact on the expansion of LSCs in regenerative medicine, especially for ocular surface reconstruction.

Purpose: To investigate the anti-proliferative effect of puerarin on retinoblastoma cells. Methods: The effect of puerarin was examined on human retinoblastoma Y79 cells using cell proliferation assays and reverse transcription-polymerase chain reaction (RT-PCR). The effect of puerarin on the cell cycle was also investigated. Western blot and RT-PCR analyses were also performed to identify the putative mechanism of action. Results: The results showed that cell viability was suppressed by puerarin in a concentrationdependent manner with a half-maximal inhibitory concentration (IC50)